The following description provides a summary of information relevant to the present disclosure and is not a concession that any of the information provided or publications referenced herein is prior art to the claimed invention.
Production of fermentation products, including ethanol, by microorganisms provides an alternative energy source to fossil fuels and is therefore an important area of current research.
The pyruvate decarboxylase and alcohol dehydrogenase enzymes of Zymomonas mobilis provide a more efficient biochemical pathway for the production of ethanol in comparison to for example the pyruvate formate lyase pathway of E. coli (FIG. 14). Dien, B. S., et al. “Bacteria engineered for fuel ethanol production: current status.” Applied Microbiology and Biotechnology 63 (2003): 258-266. The efficiency of the pyruvate decarboxylase and alcohol dehydrogenase II biochemical pathway to ethanol results at least in part from the necessary investment of only one NADH in the conversion of pyruvate to ethanol whereas other ethanol pathways such as the pyruvate formate lyase pathway require the investment of two NADH in the conversion of pyruvate to ethanol.
In the context of fermentation of the sugar xylose, ethanol production efficiency may be gained by expression of the enzyme xylose isomerase. Xylose is commonly fermented to ethanol through the intermediate xylulose-5-phosphate which is then fed into the pentose phosphate pathway. The conversion of xylose to xylulose may be a less efficient two-step conversion from xylose to xylitol then to xylulose or a more efficient one-step conversion, if xylose isomerase is present, directly from xylose to xylulose.
Vitreoscilla is a genus of filamentous gram-negative bacteria found in freshwater sediments, stagnant ponds, cow dung and decaying vegetable matter where oxygen availability is low. Vitreoscilla C1 is an obligate aerobe which synthesizes a soluble hemoglobin (VHb). VHb is a dimer of two identical subunits each having a relative mass of 15.8 kDa and a b heme. VHb is the best characterized member of the family of bacterial hemoglobin proteins.
VHb has been expressed in Saccharomyces cerevisiae (S. cerevisiae), baker's yeast, and increased ethanol production was demonstrated in comparison to non-VHb expressing controls on synthetic dextrose medium supplemented with 0.1% glucose. Chen, W., et al. “Intracellular Expression of Vitreoscilla Hemoglobin Alters the Aerobic Metabolism of Saccharomyces cerevisiae.” Biotechnology Progress 10 (1994): 308-313. Also, the VHb expressing strain was shown to grow to a lower cell culture density indicating a redirection of carbon from biomass production to ethanol. The metabolic changes to S. cerevisiae were attributed to changes in respiration and addition of respiration inhibitor antimycin A was shown to eliminate the effect of VHb on ethanol production.
VHb expression has been studied in E. coli from an isopropyl-β-D-thiogalactopyranoside (IPTG) inducible plasmid for the resulting effects on metabolism under low oxygen conditions in 0.4% glucose supplemented complex medium. Tsai, P. et al. “Effect of Vitreoscilla Hemoglobin Dosage on Microaerobic Escherichia coli Carbon and Energy Metabolism.” Biotechnology and Bioengineering 49 (1996): 139-150. It was found that increased concentrations of VHb (induced by increased concentrations of IPTG) increased the final cell culture density as measured by increases in the grams dry cell weight per liter. However, it was found that concentrations of ethanol were decreased monotonically with increasing VHb dosage. According to this study, VHb in E. coli redirected carbon away from ethanol production toward biomass production.
Most recently, VHb expression in S. cerevisiae was found to increase ethanol production efficiency on yeast synthetic complete media with 5% xylose. Ruohonen L., et al. “Expression of Vitreoscilla hemoglobin improves the metabolism of xylose in recombinant yeast Saccharomyces cerevisiae under low oxygen conditions.” Enzyme and Microbial Technology 39 (2006): 6-14. In both yeast and E. coli, xylose is metabolized to ethanol via the pentose phosphate pathway (PPP). The wild-type yeast pathway for preparation of xylose for entry into PPP comprises a two-step reductive and oxidative conversion of xylose to xylulose requiring the enzymes xylose reductase and xylitol dehydrogenase. First xylose is reduced to xylitol by xylose reductase with NADPH→NADP+ as cofactor, and second xylitol is oxidized to xylulose by xylitol dehydrogenase with NAD+→NADH as cofactor. Inefficient xylose metabolism in yeast has been attributed in part to the redox cofactor imbalance in pre-PPP xylose preparation between the reduction of xylose, which causes NADP+ accumulation, and the oxidation of xylitol, which causes NADH accumulation. One of the consequences of this imbalance is believed to be the build-up of xylitol, as low oxygen conditions limit regeneration of NAD+. In the study by Ruohonen, VHb expression was found to reduce xylitol production by as much as 40% and increase ethanol production by as much as 30%. The primary explanation for these improvements proposed by the authors was that VHb facilitated conversion of NADH to its oxidized form, NAD+, thus driving xylose from the xylitol intermediate to xylulose and thus facilitating entry of xylose into PPP.
In contrast to wild-type yeast, wild-type E. coli convert xylose to xylulose in one step with the enzyme xylose isomerase. Consequently, xylose preparation for PPP in E. coli does not have a redox cofactor imbalance and xylitol is not an intermediate. If the primary explanation proposed by the authors of the yeast VHb expression-xylose fermentation study accounts for most of the increase in ethanol production, then it would be expected that VHb would not similarly increase ethanol production from E. coli xylose fermentation.
There remains a need to develop novel microorganisms and methods which can increase the efficiency of the production of fermentation products, such as ethanol.